NIR/AIE Fluorescent Probes for Its Specific Detection Towards H₂O₂: Design Strategies, Applications and Outlook

1. Introduction

Hydrogen peroxide (H₂O₂) in the reactive oxygen species (ROS) system contributes to the cell growth and proliferation of cells [1,2,3,4]. Compared with other ROS-based derivatives, H₂O₂ has an advantage over intracellular hydrogen peroxide, which is primarily produced by the endoplasmic reticulum, mitochondria, and a variety of oxidases. Abnormal production of H₂O₂ caused Alzheimer's disease (AD), diabetes, cancer, cardiovascular disorders, and inflammatory diseases [5,6,7]. In addition to that it plays a significant role in a broad variety of pathological and physiological consequences occurred in the living organisms. Therefore, the development of near infrared (NIR) fluorescence probes for the detection of H₂O₂ can potentially contribute to human health via the use of biological applications [8,9,10,11].

NIR probes have become more important for the detection of analytes because of its unparalleled, an exceptional sensitivity, real-time imaging and visualization under the naked eye or UV light. [18,19,20,21]. As a result, there has been a rise in the development of NIR-based fluorescent probes for the precise detection of H₂O₂ in live cells and animals via the use of imaging methods. [22,23,24,25].

Development of NIR fluorescent probes usually composed of a binding site, a connecting arm and a strong fluorophore attached to it. Here, fluorophore acts as a singling unit in the NIR region. Recently, various fluorophores have been used for the design and development of NIR emission based fluorescent probes namely rhodamine, cyanine, tetraphenylethylene (TPE), triphenylamine (TPA), quinoline, boron-dipyrromethene and other aldehyde based derivatives [26,27,28,29,30,31,32,33]. The probes interaction between recognition unit and analyte depends on two different ways: i) activity based probes ii) binding based probes. In this case, the interaction depends upon the recognition unit and analytes combined with the developed NIR fluorescent probes is a common way [34,35]. In this strategy, signal to noise fluorescence imaging requires turn-on fluorescent probes that washed away repeatedly in cell imaging applications. Another strategy is key-lock interaction, which has high specificity towards target analyte in the physiological environment and can avoid impaired complexation. At the same time, if the developed fluorescent probe has a special conformation in its structure, which could avoid impairing complexation. Before complexation with target analyte, these type of probes are non-emissive in nature [36,37]. After complexation with analyte, recognition unit of the probe could enhance the fluorescence signal due to its conformational changes and structural rigidity. This review focuses on a basic design and development of NIR probes as reported by various research groups. Designing of NIR probes can be classified into three distinct methods. In type I, a fluorescent probe acts as a non-emissive in nature, which is restored by the recognition of a specific analyte. This process could be explained by the supporting of various mechanisms, such as PET, ICT and FRET processes [38,39,40,41,42].In Type-II NIR probes possess reaction sites, which are precisely triggered in the presence of target analytes followed by increase their fluorescence [43,44,45,46]. This activation takes place either by the restriction of intramolecular motion (RIM) or through the cleavage of the identifying sites accomplished through the ICT process. In type III, the probe went through a structural rearrangement as a result of the cleavage of the identifying unit, which resulted in a significant NIR emission[47,48,49].

2. Different Types of Fluorophores Used to Detect H₂O₂

2.1. Tetraphenylamine (TPA) & Tetraphenylethylene (TPE) Based Probes

Most of the TPA derivatives transmit charges and provide remarkable efficiency. Therefore, these derivatives have been used as crucial components in the area of optoelectronics. Due to the fact that high charge mobility is necessary for optimum device performance, organic field-effect transistors (OFETs) and organic light-emitting diodes (OLEDs) make substantial use of them. TPE derivatives are distinguished by their emission phenomena, which are impacted by aggregation. At larger concentrations, many fluorescent materials undergo quenching, while TPE displayed strong emission by aggregation. Because of this feature, they are widely used in sensors and bioimaging applications.

Li Fan et al. synthesized a near-infrared (NIR) fluorescent probe 1 for the specific detection of hydrogen peroxide (H₂O₂), which exhibited orange fluorescent emission [50]. Prior to the treatment, probe 1 exhibited weak fluorescence attributed to unrestricted rotation within the mito-targeting structure. Upon the addition of H₂O₂ to probe 1, the phenylboronic acid group underwent cleavage, resulting in the restricted rotation of ………. This alteration provided a strong fluorescence at 588 nm. As the concentration of H₂O₂ increased, a gradual rise in fluorescence emission was observed, followed by a color transition from colorless to an orange under UV light at 365 nm. Dong Wang et al. developed a similar NIR probe 2 for the detection of H₂O₂ using colorimetric and fluorimetric methods [51]. In colorimetric analysis, probe 2 exhibited a strong absorption band at 400 nm. Upon the addition of H₂O₂, band at 400 nm was disappeared resulting in the emergence of a new peak at 490 nm. Conversely, addition of H₂O₂ to 2, fluorescence enhancement was observed with 37.5-fold increase. a color change was observed with the naked eye and under UV light, confirming the complexation of H₂O₂ with probe 2.

Zhiqiang Mao and his team developed a Cys-triggered fluorescent probe 3 for use in complexation in the presence of H₂O₂ during apoptosis [52]. NIR fluorescent probe (add number) was selectively binding to Cys as evidenced by both colorimetric and fluorimetric titration techniques. As the concentration of Cys increased, probe 3 exhibited a strong fluorescence and saturated within 45 min. The complexation underwent additional treatment in the presence of H₂O₂, a substance frequently utilized to induce apoptosis via oxidative stress. PC12 and HepG2 cell lines were subjected to treatment with Cys in the context of an H₂O₂-induced apoptotic model. The results demonstrated that the H₂O₂-treated cells exhibited a shrivelled appearance in comparison to the control, attributed to the oxidation process of H₂O₂. The apoptotic rate increased in both PC12 and HepG2 cells during H₂O₂ incubation when treated with probe 3 in the presence of Cys. As shown in Figure 1, probes 1 and 2 did not exhibit fluorescence in HeLa cells. However, addition of H₂O₂ with varying concentration resulted in a gradual increase of the orange and yellow fluorescence. Additionally, live tissue culture was employed to identify H₂O₂ using probe 1.Tumor-containing region exhibited pronounced orange fluorescence in the H₂O₂ channel and NIR emission in the viscosity channel.

Lei Yang and their research group developed a NIR dual-channel fluorescent probe 4 for the selective detection of hydrogen peroxide, with the complexation further applied for visualization purposes [53]. Probe 4 exhibited non-fluorescence due to the photoinduced electron transfer (PET) process. Upon treatment with H₂O₂ and other ions, H₂O₂ exhibited a significant fluorescence enhancement at 550 nm. This was due to the cleavage of p-phenylboronic acid from probe 4 followed by inhibition of the PET process. A novel AIE-gen based fluorescent probe 5 was synthesized and characterized for its high responsiveness to H₂O₂ [54]. Probes were subjected to various analytes, probe 5 exhibited a significant blue-shifted fluorescence (740 nm to 525 nm) with increase of 200-fold in the presence of H2O2. Meanwhile, color transition was observed from pale green to pink. This change confirmed the binding interaction between H₂O₂ and probe 5. Additionally, the color change technique of probe 5 was applied in the RGB color assist application. Two distinct AIE-gen TPE-based fluorescent probes 6 and 7 were developed for the specific detection of H₂O₂. These probes are utilized to assess the health status of plants, identify its presence in foodstuffs, and imaging applications as reported by Shuai Zhang et al. and Lijun Tang et al [55,56]. Initially, Shuai Zhang and their research team synthesized various derivatives of TPE. All TPE derivatives exhibited strong fluorescence; however, addition of H₂O₂, a reduction in fluorescence was observed. Among the developed TPE-derived probes, only probe 6 exhibited a significant quenching effect with color change from blue to light blue. The probe 6 was subjected to testing with various analytes and organic compounds. Results indicated that probe 6 exhibited strong emission in the presence of other analytes, demonstrating its capability to selectively detect H₂O₂. Lijun Tang et al. developed a robust AIE-gen fluorescent probe for the detection of H₂O₂ in food products and bioimaging applications. probe 7 exhibited weak fluorescence. However, upon treatment with H₂O₂, there was a significant enhancement in fluorescence intensity. With an increase in the concentration of H₂O₂, there was a gradual increase in the fluorescence intensity of probe 7, accompanied by color changes from blue to pink under UV-365nm and from pale yellow to colorless in naked eye. These results attributed to the introduction of H₂O₂ to probe 7, resulting in the regeneration and restoration of the original characteristics of probe 7, as confirmed through NMR and ESI-MS. TPA and TPE based fluorescent probes were applied to various cell lines and live tissue cultures. Subsequently, probes 4 and 5 were applied to HepG2 cell lines, living mice, and zebrafish for the detection of H₂O₂ in the presence of various staining components. Probe 4 exhibited significant fluorescence emission in both the green and red channels. However, this emission was substantially quenched in the presence of H₂O₂. Notably, after pre-treatment with LPS, the robust fluorescence of probe 4 was reinstated, indicating its potential application in the treatment of inflammation in mice (Figure 2). Probe 5 underwent identical treatment protocols when applied to HepG2 cell lines and zebrafish models. Probe 5 exhibited strong emission in both the red and green channels for the HepG2 cell line and zebrafish. In contrast, addition of H2O2 showed a gradual decrease of its emission. Probes 6 and 7 were applied to plant tissue and MCF-7 cell lines. Probe 6 was applied to Orin apple calli cells, which initially exhibited a weak fluorescence in the green channel. Following the addition of NaCl stress, the green signal became apparent after the removal of the stressor. This observation confirmed that probe 6 could be applied for detecting H₂O₂ in both cells and plants. Conversely, probe 7 with MCF-7 cell lines exhibited strong emission in the red channel. Upon the introduction of HSO₃^- ions, emission was ceased entirely due to quenching effect. However, upon the subsequent addition of H₂O₂ to the same medium, emission was restored to its original intensity in the red channel. This result demonstrated that probe 7 could be effectively utilized for the detection of both HSO₃^- and H₂O₂ in cells.

2.2. Quinoline & Quinolinium Based Probes

Quinoline, a heterocyclic aromatic compound, is an excellent fluorophore due to its unique structural and electronic properties. This has made it a popular choice for developing fluorescent probes that selectively detect and bind to specific metal ions. These quinoline-based probes have been successfully employed in various applications, including detecting metal ions in food samples and biological systems. The derivatives of quinoline, with their exceptional quantum yield and high fluorescent intensity, have garnered significant interest, offering promising avenues for further research and practical applications in analytical chemistry and bioimaging. Quinolinium dyes and salts are a class of fluorescent probes that have demonstrated exceptional versatility in various medical applications. These compounds have shown significant efficacy in detecting and treating different diseases. Additionally, they have proven valuable in identifying harmful ions in food samples and provide numerous advantages for real-time monitoring applications. Designed for on-site monitoring and biological uses, Haixia Zhang and colleagues developed a new near-infrared fluorescence probe for the dual detection of hydrogen peroxide in oxidative stress and hydrogen sulfide ions [57]. Using both colorimetric and fluorimetric titration approaches, the produced probe 8 was tested with many analytes. Probe 8 showed a significant absorption band and strong fluorescence emission in their natural form as an NIR probe. Nevertheless, both techniques showed a notable new absorption peak in absorbance and a quenching impact in fluorescence emission following H₂O₂ addition. Probe 8 with H₂O₂ showed a little rise in fluorescence intensity and a drop in absorbance during the sequential investigations on complexation in the presence of H₂S. Under both techniques, H₂S produced a red shift followed by a blue to yellow color change. Lingliang Long et al. created a probe 9 for cell imaging methods and selective H₂O₂ detection in food samples [58]. In its natural form, the produced probe 9 showed weak fluorescence emission and significant absorption bands. On the other hand, a drop in absorbance and a rise in fluorescence emission in the NIR region were seen with adding H₂O₂. Moreover, an instantaneous hue shift was seen: under visible light, from red to yellow; under UV-365 nm, from pink to yellow. This result validates the development of a complex between probe 9 and H₂O₂. Based on spectroscopic data, the suggested mechanism showed that adding H₂O₂ to probe 9 caused the phenylboronic acid group from the probe's structure to cleave, hence increasing fluorescence emission. For the particular detection of H₂O₂ and viscosity in practical uses and living cells, Zhao-Min Lin and colleagues developed a FRET-based NIR fluorescent probe [59]. At 671 nm, the probe 10 displayed a red fluorescence signal which was ascribed to the FRET mechanism's suppression of the ICT process. After H₂O₂ is introduced, the red fluorescence signal at 671 nm gradually decreases while fluorescence emission at 550 nm is clearly detected. The suppression of the ICT process and the activation of the FRET mechanism explain this transformation. Without H₂O₂, two separate absorption peaks were seen in the absorbance probe 10 at 419 nm and 510 nm. After the addition, absorbance dropped at the absorption peak of 510 nm and showed a clear blue shift leading to the development of another peak at 419 nm. In both cases, a notable color shift was seen both before and after the addition of H₂O₂ with probe 10: from red to green under UV-365nm and from red to light yellow seen with the unaided eye. With rising H₂O₂ concentrations, the development in color change was essentially applied to strip paper testing. Moreover, all of these probes have been effectively used biologically on many cell lines. HepG2 cell line and zebrafish were used for Probe 8 evaluation in order to detect H₂O₂. Probe 8 showed red and yellow fluorescence emission both in and absent of H₂O₂. Strong red fluorescence in the NIR channel allowed Probe 9 to be applied to SW1783 cells for an endogenous detection of H₂O₂. But when H₂O₂ was added, the red emission in the NIR channel dropped greatly while the green channel showed a high fluorescence emission. Furthermore, probe 9 was used very well for the H₂O₂ detection among many food samples as seen in Figure 3. On pre-incubation, Probe 10 was treated with HeLa cells in the absence of TEMPO (H₂O₂ scavenger), therefore reducing fluorescence emission attributable to the removal of TEMPO by endogenous H₂O₂. The experiment was carried out under H₂O₂ presence, incubated with TEMPO and endogenous H₂O₂. The fluorescence intensity ratio seen throughout the red/green channel then changed as a consequence. The experimental data showed that probe 9 can identify both foreign and endogenous forms of H₂O₂.

For the aim of monitoring H₂O₂ in diabetes, different cancer cell lines, and mouse models, the construction of quinolinium-based NIR fluorescent probe 11 was performed [60]. In investigations incorporating both absorption spectra and fluorescence spectra, the created probe 11 was treated with cations, anions, enzymes, and amino acids among other analytes. Probe 11 showed a significant absorption peak before different analytes were introduced. But the separate peak faded as the concentration of H₂O₂ rose, creating a new peak. Probe 11 showed modest fluorescence emission in the fluorimetric method under 725 nm of excitation wavelength. Fluorescence emission of the probe tested in isolation against the same analytes showed no variations. But adding H₂O₂ produced a slow rise in fluorescence intensity at 772 nm. Intramolecular charge transfer (ICT) mechanism causes the change in fluorescence emission. The ICT process was limited before H₂O₂ was developed, so fluorescence did not exist. But adding H₂O₂ caused the ICT process to be inhibited, which triggered fluorescence and increased fluorescence intensity. Probe 11 consists of a hydroxyl unit functioning as a fluorophore agent overall. This unit is released upon H₂O₂ addition, and strong fluorescence signal arises from borate ester group consumption. Wenbin Zeng and colleagues created a red-light emissive fluorescent probe using quinolinium as a fluorophore for the particular detection of hydrogen peroxide in live cells and rheumatoid arthritis. Successful characterization of the created probe 12 using colorimetric and fluorimetric titration approaches preceded its examination against different metal ions [61]. Original version of 12 showed absorption peak at 425 nm as an AIE probe. H₂O₂ caused this peak to vanish and a new absorption peak at 400 nm to arise. Probe 12 showed modest fluorescence emission in photoluminescence spectra ascribed to the central double bond. But when H₂O₂ was added, fluorescence emission was considerably increased, especially at 620 nm. The suggested mechanism is that a tetrahedral boronate intermediate results from H₂O₂ interacting with the electrophilic boron atom in the phenylboronate of the probe. Spectroscopic methods verifying the binding of H₂O₂ to probe 12 support this interaction even further. Using a new Aβ-targeted and blood-brain barrier (BBB)-permeable ratiometric H₂O₂-responsive fluorescence probe 13, Hung-Wing Li and colleagues highlighted its application benefits in cellular and murine models of Alzheimer's disease [62]. At 388 nm, Probe 13 showed a wide absorption peak; at 574 nm, it had a modest emission peak. But the addition of H₂O₂ produced a red shift in the weak emission peak and the absorption peak to 485 nm and 661 nm correspondingly. Whereas the fluorescence intensity of the probe dropped at 574 nm in the fluorimetric titration method at an excitation wavelength of 490 nm, emission gradually rose at 661 nm. The production of the oxidized product came from 1,6-elimination via the methylphenyloxy linker in the phenolic intermediate increasing fluorescence emission. Furthermore, NMR and ESI-MS spectroscopic methods were used to confirm the oxidizing within the structure of the probe, therefore supporting the binding stoichiometry. Reportedly designed for selective administration to mitochondria, a fluorescent probe assigned as 14 showed extraordinary selectivity for H₂O₂ in brain tissue [63]. The probe 14 showed in the UV-visible spectra a clear absorption peak at 560 nm in water that changed to 590 nm in glycerol. Under fluorescence titration at 440 nm excitation wavelength, the fluorescence intensity showed a notable increase of around 120-fold at an emission wavelength of 670 nm. Colorimetric probe 14 showed an absorption peak at 420 nm when H₂O₂ was added; this is ascribed to the binding stoichiometry between probe 14 and H₂O₂. Adding H₂O₂ in fluorimetric analysis produced a notable increase in fluorescence intensity, by a factor of 80 from the first measurement. Following the addition of H₂O₂, the oxidation and hydrolysis processes in the probe's phenylboronic acid group produced changes in fluorescence intensity that later results in elimination via intramolecular rotation. Spectroscopic methods and DFT analysis tracked the reaction's development to show that H₂O₂ bound to the complexation of probe 14. Consistent use of confocal and fluorescent microscopy imaging methods allowed the remaining probes for biological studies to be done as shown in Figure 4. Examined against HeLa cell lines, probes 11 and 12 showed modest fluorescence emission in the red channel. Increasing amounts of H₂O₂ improved this emission. Using both non- and neuronal cell lines—more especially, N2a and N2aSW— Probe 13 was investigated. A notable fluorescence emission in the red channel came from increasing the H₂O₂ concentration. In cell imaging, the probe 14 experiment was carried out using SH-SY5Y human-derived neuroblastoma cell line. With yellow fluorescence, the probe showed modest emission; this effect changed with increasing H₂O₂ concentration. By use of a strong zone instead of weak yellow fluorescence emission, increasing the quantity of H₂O₂ confirmed the identification of H₂O₂. In a mouse model, the probes 13 and 14 were efficiently used to show a notable active fluorescence response following H₂O₂ injection in the pre-treated mouse model with probes 13 and 14.

2.3. Pinocol Based Fluorescent Probes

Pinacol derivatives are essential in the development of probes, serving as adaptable functional handles and strong structural scaffolds. Their distinct characteristics enable the alteration of fluorophores, the addition of protecting groups, and the formation of bioconjugation linkers. Their role is crucial in the development of reactive oxygen species (ROS) probes, as they initiate a fluorescent signal upon cleavage by H₂O₂ or other ROS. Additionally, pinacol improves the stability and solubility of boronic acids during probe synthesis, thereby increasing their effectiveness. Pinacol-like diols demonstrate the capacity to bind analytes via boronic acid-diol interactions, which is beneficial for the accurate detection of glucose and fructose. The future of this field presents significant potential, incorporating two-photon probes for deep-tissue imaging, AIE probes for amyloid detection, and advanced metal complexation methods for hypoxia sensing. These derivatives, particularly boronate esters, play a vital role in the selective detection of reactive oxygen species (ROS) and other biological analytes. Their application significantly contributes to advancements in imaging technologies and improves our comprehension of intricate biological systems. To ensure low interference from other analytes, Tongsheng Chen and colleagues developed a near-infrared emitting ratiometric fluorescence probe intended for the selective detection of hydrogen peroxide [64]. The designed probe 15 proved to be very useful in cell imaging techniques for the detection of H₂O₂. First, the water-soluble ESIPT-based NIR fluorescent probe was tested using many analytes using both colorimetric and fluorimetric titration methods. The probe in colorimetric titration showed a significant absorption peak at 365 nm. But when H₂O₂ was added, the absorption peak at 365 nm shifted somewhat to 422 nm. In fluorimetry, without H₂O₂, excitation at a wavelength of 422 nm produced the probe displaying an enol emission at 538 nm. At 656 nm, we saw a notable red-shift in fluorescence emission. The observed red-shift in absorbance and fluorescence emission with H₂O₂ addition points to complex development. Furthermore seen was a color shift from colorless to yellow visually and from colorless to pink under UV-365 nm light with the addition of H₂O₂ to probe 15. Using an intracellular approach, a new NIR fluorescent probe 16 was designed for hydrogen peroxide detection and visualization [65]. Following the determination of the excitation wavelength, probe 16 was assessed under different cations, anions, and amino acid concentration. Attributed to the limitation of the intramolecular charge transfer (ICT), the probe showed mild fluorescence emission and no spectrum alterations together with other metal ions. H₂O₂ added to probe 16 produced a notable increase in fluorescence emission in the near-infrared spectrum along with a considerable Stokes shift of 122 nm. H₂O₂ added to the near-infrared emission produced a hydroxy group from the structure of the probe. To specifically detect H₂O₂ in melanoma, Guorui Li and colleagues created a dual locked NIR fluorescent probe using methylene blue. Cell lines and animals with tumours were used for application investigations [66]. Developed using methylene blue, probe 17 showed a turn-off fluorescence characteristic and modest fluorescence emission. Probe 17 shown absorption peak at 665 nm in colorimetric titration. It showed significant fluorescence in fluorimetric titration at 684 nm excitation wavelength. Probe 17 is used in combination with borate to detect H₂O₂, therefore enabling a turn-off to a turn-on fluorescence state. This procedure produces a 17-fold rise in fluorescence intensity following emission enhancement. The increase in emissions came from H₂O₂ transforming a non-fluorescent probe via cascade activation, hence activating the fluorescence signal from an "off" state to "on" one. As we seen in Figure 5, applications in both endogenous and exogenous cellular imaging, a new artificial intelligence fluorescent probe has been designed to see H₂O₂.

Using imidazo pyridine conjugation, Junbing Jiang and colleagues created probe 18 that shows improved fluorescence for the detection of H₂O₂, hence reducing interference from other analytes [67]. Probe 18 shown low absorbance and fluorescence emission in both colorimetric and fluorimetric methods. But when stimulated at 500 nm, adding H₂O₂ produced a boost in fluorescence at 350 nm. Under UV-365nm light, a color change from blue to yellow was seen along with a matching slow rise in fluorescence intensity when the concentration of H₂O₂ was raised. The noted fluorescence increase and color shift demonstrate H₂O₂'s binding recognition to probe 18. Designed and developed for the dual detection of H₂O₂ and HOCl, a bifunctional fluorescent probe designated as 19 Shunping Zang et al. Analyzed utilizing colorimetric and fluorimetric approaches for the detection of H₂O₂ and HOCl across two different channels, the bifunctional probe assigned number 19 showed two different absorption peaks [68]. Probe 19 showed an absorption peak at 480 nm; yet, a little red shift in the absorption peak was seen when H₂O₂ was added, ranging from 480 nm to 455 nm. Probe 19 showed weak fluorescence intensity in the absence of H₂O₂ when the excitation wavelength was set at 600 nm in the fluorimetric titration technique. On the other hand, in the presence of H₂O₂ the fluorescence emission showed an increase in intensity at 685 nm; other metal ions, in combination with probe 19, showed no changes in fluorescence intensity. Together with a little red shift in both methods, the addition of H₂O₂ causes changes in fluorescence emission and absorption, therefore indicating the development of a complex in red fluorescence. Particularly aiming at the detection of H₂O₂, the use of pinacol ester-based fluorescent probes was effectively carried out for cell imaging and biological uses. Treatments involving MCF-7 cells, HeLa cells, and RAW 264 cells applied probes 15, 16, and 19. In the experiment including seven cells and zebrafish for the H₂O₂ imaging, it was found that in the absence of H₂O₂, neither the red nor green channels showed emission. On the other hand, in the presence of H₂O₂, the red and green channels showed a great activity. While tumor-bearing mice were subjected to H₂O₂, pre-treatment for the probes 17 and 18 consisted of A375 cells and A549 cells. Strong fluorescence emission was shown by the cell lines pre-treated with probes 17 and 18; correspondingly, probe 17 displayed red channel fluorescence and probe 18 displayed green channel fluorescence (Figure 6).

2.4. Aldehyde Based Fluorescent Probes

Because of their great reactivity and broad range of use, Aldehyde-based fluorescent probes are very vital in chemical and biological research. These probes have great reactivity thanks to aldehyde groups (–CHO), which may be exactly changed to recognize a wide spectrum of analytes including thiols, Reactive Oxygen Species (ROS), Reactive Nitrogen Species (RNS), and amines/amino acids. In response to specific target molecules, these sophisticated fluorescent probes show amazing capacity to either activate (turn-on) or deactivate (turn-off) fluorescence. This special ability helps scientists to precisely monitor biological events. In fields like biochemistry, medicine, and environmental research, aldehyde-based fluorescence probes have become indispensable tools. Their great selectivity, sensitivity, and adaptability make them useful instruments for the detection of important analytes and help to reduce interference. Moreover, their ability to provide real-time, non-invasive imaging sets them as essential instruments in the development of our knowledge of disease processes and the enhancement of diagnostic capability. By means of these probes, scientists may get important insights that propel development in fundamental science and clinical applications, hence generating major discoveries in health and environmental sustainability. Kehua Xu and associates have created a new mitochondria-targeting NIR fluorescent probe 20 to clarify how H₂O₂ and other metal ions affect viscosity interactions [69]. Under low viscosity, the probe 20 displayed modest fluorescence emission because to molecular rotation. On high viscosity, on the other hand, the molecular rotor is hindered and probes 20 exhibits intense fluorescence emission. Using an excitation wavelength of 685 nm, the fluorimetric titration technique for probe 20 was assessed with respect to other metal ions and H₂O₂. Under low viscosity circumstances, the molecular rotor showed mild fluorescence emission; this was improved upon H₂O₂ addition. H₂O₂ produced the probe 20 with high fluorescence emission, which is characterized by an intensity rise. Still, too strong metal ions might cause viscosity to drop. The state of probe 20 concerning viscosity shows the stability of the probe under different circumstances. Furthermore, probe 20 might be a useful fluorescent agent for observing changes in mitochondrial viscosity in physiological surroundings. Designed for use in cell imaging and food sample monitoring of HSO₃^- and H₂O₂, a reversible near-infrared fluorescent probe under designation 21 was created. Qingtao Meng and colleagues created a probe with a D–π–A–π–D conjugating structure combining a benzopyrylium moiety with a C=C double bond [70]. First testing the probe 21 used cations, anions, and amino acids among other analytes. The findings showed that in contrast to other metal ions, the probe specifically picks up HSO₃^- ions. Probe 21 detects HSO₃^- ions only in absorbance spectra, displaying a red-shift-oriented new absorption peak. Moreover, the Michael addition process disturbs the link between the C=C double bond and the benzopyrylium moiety, therefore progressively reducing another absorption peak at 605 nm. In fluorimetry, probe 21 detects HSO₃^- ions by noting a decrease in fluorescence intensity, ascribed to quenching of fluorescence emission with respect to other metal ions. Variations in the probe's moiety after HSO₃^-) introduced explained the variations in absorbance and emission. Visually, one also saw a hue shift from blue to colorless. Sequential sensing of H₂O₂ using the complexation of probe 21 with HSO₃^- resulted in the fascinating recovery of the spectroscopic characteristics of probe 21. The main absorption peak at 605 nm shows a rise in concentration of H₂O₂; the peak almost returns to its former location. Noted with increasing H₂O₂ concentration during complexation, the observed color shift moves from colorless to blue. This obviously shows that the recycling procedure may help to recover the spectroscopic characteristics of probe 21 in the presence of H₂O₂. Developed for selective sensitivity to ROS/RNS and base ions, a dual responsive NIR-II fluorescent probe 22 may be rather important in biosensing applications [71]. Development and efficient usage of the NIR-II probe for biosensing purposes in a mouse model to track cystitis and colitis under different pH levels and time intervals is discussed (Figure 7). Evaluated with many analytes, the produced probe 22 showed emission in the NIR-II spectrum. The probe showed an absorption peak at 870 nm; treatment with H₂O₂ produced a red shift of the absorption peak. Probe 22 shown mild fluorescence emission in fluorimetric titration. From 0 to 500 µM, a rise in the concentration of H₂O₂ produced a slow increase in fluorescence intensity accompanied by a little red shift from its natural location. The limit of the intramolecular charge transfer (ICT) mechanism before the introduction of H₂O₂ explained the sudden changes in absorbance and emission. The hydroxyl group was eliminated upon H₂O₂ addition, producing a phenolic fluorophore effect displaying the ICT process. Designed for the evaluation of the effectiveness of photodynamic and photothermal synergistic treatment in cell imaging applications, Chun- Yan Li and the research team have developed a dual activated NIR-I/NIR-II fluorescent probe. Designed and constructed, the probe 23 showed a mild fluorescence effect, hence absenting photodynamic treatment (PDT) and photothermal therapy (PTT) [72]. The probe 23 was investigated both in the presence and absence of H₂O₂ with viscosity changes meant to improve the fluorescence effect. Under high viscosity, the probe showed two absorption peaks in colorimetric analysis at 725 nm and 798 nm. On the other hand, the absorption peaks were strengthened when high viscosity was maintained in the presence of H₂O₂. Under high viscosity, probe 23 shown no fluorescence response at wavelengths of 810 and 945 nm in the fluorimetry study. Under the same high viscosity circumstances, however, the addition of H₂O₂ produced a notable fluorescence response marked by a fluorescence intensity increase. Further confirming that probe 23, which showed weak fluorescence, may be triggered only in the presence of H₂O₂ under circumstances of high viscosity, are the studies using colorimetric and fluorimetric approaches. Selective detection of H₂O₂ utilizing both colorimetric and fluorimetric techniques made use of another AIE fluorescent probe. Its use was shown using live tumor cells and in-vivo experiments [73].

Originally combined as a solution comprising probe 24 and H₂O₂, the produced probe showed a significant peak at 430 nm. But a new strong absorption peak with a little blue shift at 450 nm emerged during incubation with H₂O₂. Probe 24, which included H₂O₂, first showed quenched fluorescence emission after the excitation wavelength for fluorimetric titration being adjusted at 450 nm. After adding H₂O₂ during incubation, this emission was then strengthened and shown great fluorescence at 630 nm. The addition of H₂O₂ to the probe 24 + H₂O₂ combination split the borate moiety from the probe, therefore producing changes in emission and absorbance. This mechanism enabled the AIE fluorophore to be released, hence increasing fluorescence intensity and fluorescence activation. Designed for dual detection of SO₂ and H₂O₂ in conditions characterized by low polarity and high viscosity, Jun Ai and colleagues have developed a flexible fluorescent probe 25 [74]. Attractive near-infrared characteristics for polarity, viscosity, and SO₂/ H₂O₂ levels allowed the probe 25 to show a clear fluorescence emission peak at 705 nm. Probe 25 showed highest absorption in the absorbance spectra at 648 nm, which matched the excitation wavelength of 648 nm. With a progressive concentration, the fluorescence emission at 705 nm showed a notable drop in fluorescence intensity. On the other hand, the fluorescence intensity at 705 nm emission also showed rise when viscosity rose progressively. SO₂ produced a decrease in the absorption peak at 648 nm and the emission peak at 710 nm when testing probe 25 with SO₂/ H₂O₂. Sequential sensing in the presence of H₂O₂ restored both the absorption peak and emission peak to their natural forms. Further evidence of complicated development came from the color change from blue to colorless in the presence of SO₂ reversing from colorless back to blue in the presence of H₂O₂. An uncontrolled intramolecular charge transfer (ICT) activity in probe 25 explains the observed variations in absorbance and emission. Under low polarity and high viscosity especially, the twisted intramolecular charge transfer (TICT) mechanism completely reduced this activity in presence of SO₂/ H₂O₂. Using a similar approach considering polarity and viscosity parameters, a second NIR fluorescent probe 26 was developed specifically for the detection of H₂O₂ across different cell lines [75] (Figure 8). Under high viscosity, the probe in a free condition showed a pronounced absorption peak at 585 nm, which was much rose at the same wavelength. High viscosity conditions in fluorimetry produced a notable increase in fluorescence emission at 750 nm along with a considerable Stokes shift of 165 nm. Unlike polarity, the fluorescence emission of probe 26 was much reduced by an 82-fold. After polarity and viscosity experiments, probe 26 was assessed in both colorimetric and fluorimetric environments in presence of H₂O₂. Probe 26 showed a clear absorption peak at 560 nm, which then reduced following the addition of H₂O₂ to produce a new absorption peak at 405 nm. In fluorimetry, probe 26 first showed faint emission. H₂O₂ added raised the fluorescence intensity by a factor of 20 at 650 nm. Moreover, the addition of H₂O₂ to probe 26 clearly changed the color from purple to yellow, suggesting the development of a complex. The discussed probes here were successfully utilized for the detection of H₂O₂ under different viscosity and polarity against HepG2 cells (20), Zebra and mouse (21), mice (22), HeLa cells and tumor bearing mice (23), 4T1 cells and 4T1 bearing mice (24), HeLa and CT26 cell lines (25) and 4T1 cells and mice model (26).

2.5. Hemicyanine Based Fluorescent Probes

Because of its amazing characteristics—including improved quantum yields and configurable spectrum features fluorescent probes based on hemicyanine are becoming more and more common in scientific teaching and research. Their near-infrared (NIR) spectrum of absorption and emission greatly increases their relevance in disciplines like bioimaging, medical diagnostics, and real-time biomolecule identification. High-resolution imaging in complicated biological contexts depends on the probes penetrating deeply into biological tissues and simultaneously maintaining a suitable signal-to----noise ratio. Recent developments in hemicyanine dyes have produced imaging probes intended especially for targeting mitochondria. The probes show better endurance and increased fluorescence qualities. Currently, research efforts concentrate on improving these probes to maximize their biocompatibility and solve any limits for in vivo uses. Designed for the imaging and detection of H₂O₂ in many disease types, including lung, liver, and cancers, a flexible NIR fluorescent probe 27 was created [76] (Figure 9). Weak absorption and emission in the NIR range were ascribed to the limitation of the intramolecular charge transfer (ICT) mechanism within the probe, hence quenching of fluorescence. Apart from a lesser absorption peak at 698 nm, the probe 27 showed two clear absorption peaks at 546 nm and 576 nm. The absorbance of the peaks at 546 nm and 576 nm dropped as the quantity of H₂O₂ rose; the absorbance at 698 nm rose dramatically. Probe 27 was tuned in fluorimetric titration at 698 nm. Along with a color shift from purple to light blue, the probe showed modest emission at 720 nm that considerably increased with increasing H₂O₂ concentration from 0 to 500 µM. The uncontrolled intramolecular charge transfer activity, which triggered the fluorescence, changed absorbance and emission with the addition of H₂O₂. Designed for the particular detection of H₂O₂, a fluorescent probe 28 shows a fast and selective response in detecting H₂O₂ within active tumor cells [77]. Under the presence and absence of H₂O₂, the photophysical response of probe 28 was evaluated using UV-visible absorption and fluorescence spectroscopy. Under UV-visible spectrum analysis, probe 28 showed a large absorption peak at 584 nm. H₂O₂ caused this peak to move to 681 nm and a color change from violet to blue. Probe 28 showed little fluorescence emission in fluorimetric titration spectra when stimulated at 702 nm. With increasing H₂O₂ concentration, this emission was much raised, almost 25-fold. The complexation solution was incubated under many biomolecules both in presence and absence. The findings showed that the other biomolecules did not interfere with the complexation, therefore verifying the relevance of probe 28 for biological uses in the selective detection of H₂O₂. Aimed at monitoring in plants, Na Niu and colleagues developed a new NIR fluorescent probe for the particular detection of H₂O₂ [78]. Examined under many biomolecules in combination with H₂O₂ at an excitation wavelength of 650 nm, the built probe 29 showed 29 was treated with H₂O₂ both in and without of the reagent as an NIR probe. Excitation at 650 nm produced modest emission at 720 nm in the absence of H₂O₂; this was much improved with the addition of H₂O₂. Probe 29 was assessed in the presence of H₂O₂ throughout a pH range of 3 to 10 in order to improve the fluorescence intensity even further. Still, the probe showed no change in emission in both acidic and basic environments. Probe 29 showed a slow rise in fluorescence intensity under neutral and alkaline conditions in the presence of H₂O₂ along with a little red shift in emission ascribed to the intramolecular charge transfer (ICT) mechanism. Combining a boronic acid ester as a recognition site for hydrogen peroxide (H₂O₂) and biotin as a tumor identification site. Probe 27 first was used for the H₂O₂ detection on HeLa cells as well as liver, lung, and animal models. Probe 28 then was used to mouse tumor tissue and A49 cells. Analyzed utilizing many plant components, including leaves and onions, Probe 29 showed a high red emission in response to fluorescence imaging detection of H₂O₂.

Hong Liu and colleagues created a near-infrared fluorescence probe This probe may increase fluorescence emission in reaction to H₂O₂ presence [79]. In in vivo applications, the created probe 30 was used quite well to detect H₂O₂. Using absorbance and emission spectroscopic analyses, Probe 30 was investigated both in presence and absence of H₂O₂. The probe in its natural form had a significant absorption peak at 544 nm in UV-visible spectra. But when H₂O₂ was present, the absorption peak shifted from 544 nm to 680 nm and the hue changed from purple to blue. Fixing the excitation wavelength for fluorimetric titration, the feeble emission of the probe at 707 nm showed a slow rise in intensity with increasing H₂O₂ concentration. As the concentration of H₂O₂ was raised gradually from 0 to 100 µM, the emission changed. Especially, fluorescence intensity increased significantly at a dosage of 50 µM. The interaction between the boronic acid ester group and H₂O₂ explains this rise in fluorescence emission in the near-infrared (NIR) region. The complicatedation of probe 30 with H₂O₂ was assessed with respect to other reactive oxygen species (ROS) molecules concurrently. There was no change in fluorescence intensity, hence it is clear that probe 30 might be rather important for the selective detection of H₂O₂ in practical uses. For the intended imaging of mitochondrial H₂O₂ in the presence of viscosity, Yang Liu and colleagues have developed a dual-modal probe, NIRF/PA [80]. Apart from in vivo and in vitro uses aiming at targeting mitochondria and imaging changed H₂O₂ levels and viscosity inside cells, the developed probe 31 was efficiently used for the diagnosis of PD. First used in absorbance spectra, the special probe 31 showed a significant absorption peak at 710 nm. A little change in fluorescence intensity was seen when the excitation wavelength was set at 725 nm and the concentration was raised from 0 to 200 µM. The probe was then tested against glycerol at different concentrations ranging from 0 to 90%, where a significant increase in intensity was seen. Examined under many situations, Probe 31 included glycerol and H₂O₂ both present and absent. A modest increase in emission and absorption was seen in the presence of H₂O₂/glycerol. On the other hand, simultaneous application of H₂O₂/glycerol clearly increased fluorescence intensity and absorbance. Other analytes then were investigated under the H₂O₂/glycerol system, where no interference from other analytes was noted. Under the presence of H₂O₂, the borate unit in probe 31 experienced an oxidative elimination process that restored fluorescence. DFT, FT-IR, and ESI-MS spectroscopic methods were used to verify the complexation system and prove the oxidative elimination process took place within the probe. Using colorimetric and fluorimetric techniques, Bo Tang and colleagues developed Probe 32 for the particular detection of H₂O₂ and viscosity [81]. For in situ H₂O₂ detection as well as in vivo viscosity biological uses, the probe 32 proved really useful. Probe 32 was tested first using many analytes. Whereas the other analytes did not show any further spectrum changes, the addition of H₂O₂ changed absorbance and fluorescence emission. Applying the same method, the probe's viscosity response was assessed. It was seen that the absorbance and emission of probe 32 likewise rose when the viscosity concentration rose. Structural alterations cause a slow reduction in fluorescence emission when H₂O₂ is added to the 32+viscosity system. In low viscosity, however, probe 32's fluorescence emission is modest; so, under high viscosity circumstances, it recovers to its natural form. The spectrum investigations demonstrate that probe 32 is able to detect viscosity and H₂O₂ selectively in biological applications spanning many excitation wavelengths. For the detection of H₂O₂ in different cancer cell lines and mice models, all under mentioned probes were efficiently used. While probe 31 was assessed using SH-SY5Y cells and Drosophila culture (Figure 10), where it demonstrated high red fluorescence emission, probe 30 underwent successful testing with 4T1 cells and a 4T1 tumor-bearing animal model. In tests involving mice and foam cells, Probe 32 was used to identify H₂O₂ via confocal fluorescence imaging.

2.6. Isophorone Based Fluorescent Probes

As the field of chemical and biological sensing continues to advance, the examination of fluorescent probes based on isophorones has become an extremely significant area of study. It's possible that this transformation is connected to the vast range of applications that these probes have, in addition to the excellent photophysical properties that they possess. It is possible to give an inherent value to fluorescent probes due to the fact that they emit light, are sensitive to changes in the environment, and have outstanding versatility in applications linked to chemical and biological sensing. The capability of these technologies to deliver high-contrast imaging in real time positions them as key instruments in a number of different fields, including biology, environmental monitoring, and materials research, among others. It seems from this that there is potential for significant advancements in both research and application. Using dicyanoisophorone and dicyanomethylene derivatives for the particular detection of H₂O₂, three different research groups have created and produced NIR-based fluorescent probes 33 [82], 34 (i), 34 (ii) [83], and 35 (i), 35 (ii) [84]. Every probe has been used successfully in a biological context. Probe 33 was first checked under many analytes. Red shift-driven notable changes in absorbance and fluorescence emission were seen with the addition of H₂O₂. The removal of the phenyl boronate group in the fluorophore caused the variations in fluorescence absorption and emission; this increases fluorescence intensity in the near-infrared range. Treating Probes 34 (i) and 34 (ii) with H₂O₂ during a 0 to 240 minute period resulted in Whereas probe 34 (ii) finished the procedure in 87 minutes, showing a quicker reaction than probe 34 (i), probe 34 (i) needed 240 minutes for the treatment. Examined in the presence of H₂O₂ throughout a range of concentrations from 0 to 100 equivalents was Probe 34 (ii). With an excitation wavelength set at 550 nm, the data revealed a slow rise in fluorescence intensity at 645 nm. Examining probe 34 (ii) behaviors in the presence and absence of other metal ions alongside H₂O₂ allowed one to assess interference from other analytes. The findings showed that during the complexation of probe 34 (ii) with H₂O₂ no interference from other metal ions took place. The addition of H₂O₂ caused the changes shown in the emission spectra; this helped the borate group to cleave off the fluorophore, hence improving the fluorescence intensity. The probe showed an ICT process limitation before the addition of H₂O₂, which was then inhibited after the reaction with H₂O₂. Using dicyanomethylene, two NIR fluorescent probes—35(i) and 35(ii)—have been created for the particular detection of H₂O₂ across many cell lines and in mice within the NIR range. Prior to and during the addition of H₂O₂, photophysical tests of the probes were performed using both colorimetric and fluorimetric methods. Along with absorption peaks at 410 nm and 413 nm before the addition of H₂O₂, the probes 35(i) displayed modest emission at wavelengths of 653 nm and 655 nm. Under H₂O₂, a slow rise in fluorescence emission was seen along with a significant absorption peak cantered at 410 and 412 nm. The fluorescence emission at 653 nm showed a slow rise in intensity when one raised the concentration of H₂O₂ from 0 to 100 µM. Tested against many analytes, the complexation technique showed no interference from other analytes—which would be important for biological uses. As well as MCF-7, MCF-10A, DU-145 cells, and tumor-bearing mice for probes 35(i) and 35(ii), the dicyanoisophorone and dicyanomethylene based probes presented in this discussion were essentially used for the detection of H₂O₂ across various cell lines, including HepG2 cells, zebrafish, and inflammatory mice models for probe 33; HeLa cells and zebrafish for probes 34(i) and 34(ii). The probes showed mild fluorescence emission across many channels; nevertheless, the addition of H₂O₂ produced significant fluorescence emission in the red channel (Figure 11).

2.7. Indole Based Fluorescent Probes

Indole derivatives are key components of fluorescent probes due to their photophysical properties, structural flexibility, and ability to interact with a wide range of analytes. These precise probes are essential for detecting metal ions, anions, ROS/RNS, pH alterations, biomolecules, and cellular microenvironments. Indole derivatives are effective in identifying and quantifying analytes due to their strong fluorescence emission and high quantum yields. Many indole derivatives exhibit a large Stokes shift, reducing self-quenchishing and improving signal-to-noise ratio. This trait is crucial for reliable detection in complex environments with interference. Indole derivatives employ Intramolecular Charge Transfer (ICT) and Photoinduced Electron Transfer (PET) to detect metal ions, anions, and reactive species (ROS/RNS). The techniques increase sensitivity and specificity, allowing precise measurement of trace analyte levels. Indole-based fluorescent probes are useful for biological monitoring of biomolecules, cellular processes, and physiological conditions. Biosensing technologies and medical diagnostics have evolved due to contributions, allowing inventive healthcare methods. Fluorescence probes need indole derivatives' particular properties and versatility, enabling substantial advances in many scientific fields and valuable applications. Based on indole, three independent research teams have produced three different NIR-I/II fluorescent probes for the particular detection of H₂O₂ using spectroscopic investigations. Crucially for cell imaging research, Hu Xiong et al. created an NIR-II fluorescent probe 36 with great selectivity and sensitivity towards H₂O₂, thereby enabling a change in fluorescence emission from turn-off to turn-on [85]. Analyzed in its present state, Probe 36 showed an absorption peak at 525 nm and a faint emission at 990 nm, ascribed to the development of an acceptor-π-acceptor (A-π-A) system. From 0 to 250 µM, the addition of H₂O₂ causes variations in absorbance and emission. Whereas another absorption peak at 790 nm indicated a rise in colorimetric analysis, the absorption peak at 525 nm showed a decline. By contrast, the fluorimetric study found a notable Stokes shift of 200 nm and a marked increase in the fluorescence emission peak at 990 nm. The modifications were ascribed to the inclusion of H₂O₂, which experiences structural rearrangement in concert with a donor-π-acceptor (D-π-A) system, hence generating NIR-II fluorescence emission via an ICT process. David G. Churchill and colleagues created a highly selective NIR fluorescent probe called 37 for sensitive detection of H₂O₂ in the presence of several ROS and RNS analytes by use of an ICT sensing system [86]. Without analytes, the probe 37 in a free form was investigated and showed a significant absorption peak at 527 nm that changed to 552 nm following H₂O₂ addition. Furthermore, the free-state probe 37 showed modest fluorescence emission at 617 nm with the excitation wavelength set at 552 nm; this effect grew with increasing H₂O₂ concentration from 0 to 18 equivalents. No interference was seen in the complexation state of probe 37 with H₂O₂; during complexation, the presence of certain reactive oxygen species (ROS) and reactive nitrogen species (RNS) analytes did not produce any changes in emission. The presence of H₂O₂, which serves as an electron receptor and helps the ICT process from the indole nitrogen donor inside the probe structure to the malononitrile ring, was responsible for the noted variations in emission and absorbance measurements. For the particular detection of hydrogen peroxide (H₂O₂) in tumor targeted uses for cell imaging, Wancun Zhang and colleagues created a new near-infrared fluorescent probe using indole derivatives [87]. Two absorption peaks at 600 nm and 660 nm on the produced probe 38 showed a significant change from 600 nm to 686 nm following H₂O₂ injection. Probe 38 showed a modest fluorescence signal in its natural form at 710 nm in the emission spectra. Nevertheless, the fluorescence emission at 710 nm exhibited a slow rise in intensity when the concentration of H₂O₂ was raised from 0 to 100 µM, culminating at 100 µM. The suppression of the ICT process caused by the replacement of the phenyl borate ester group with the hydroxyl group of the probe produced the emission and absorbance seen before the addition of H₂O₂. H₂O₂ brought about changes in absorbance and an increase in emission. The cleavage of the phenyl borate group is responsible for this alteration as it generates a strong NIR fluorescence signal by releasing a hydroxyl group. Strong fluorescence emission in the presence of H₂O₂, which was essentially used for the detection of H₂O₂ in biological applications, shown by the probes produced from indole derivatives. Using an APAP-induced liver damage mice model and a HeLa cell line, the probe 36 was assessed to detect H₂O₂ using fluorescent NIR-II imaging method (Figure 12). Pre-incubated under probe 36, the cells and mouse model were investigated both with and without H₂O₂. Imaging showed little emission in the absence of H₂O₂; significant NIR-II fluorescence signals were seen in the presence of H₂O₂. Under the same technique, a further probe assigned number 37 was tested against the lung cancer cell line (A459 type) and zebrafish. Using H₂O₂ produced a strong fluorescence signal seen in the red emission channel across both models. Examined in both presence and absence of H₂O₂ inside the 4T1 cell line at different concentrations was probe 38. H₂O₂ caused a significant fluorescence signal in the red emission channel. NAC addition helped the fluorescence signal to return to its natural condition.

2.8. Chromene Based Fluorescent Probes

Chromene is a crucial chromophore for creating new dyes with aggregation-induced emission (AIE) properties, specifically for near-infrared (NIR) applications in living organisms. Its significance lies in developing innovative dye structures and modifying existing ones for various uses. These include photoacoustic imaging, photothermal therapy, metal ion sensing, and drug delivery. Key objectives drive the design and modification of these dyes: creating novel crossbreeding skeletons to enhance dye performance, broadening the wavelength range for improved functionality, ensuring structural stability, and implementing strategies to increase brightness and overall efficacy. A chromene based NIR fluorescent probe was designed and examined for the sequential detection of H₂O₂ using SO₂ complexation with probe 39 [88]. As a colorimetric probe, 39 was first tested against different metal ions were there was an strong absorption peak for probe alone at 650 nm which was completely decreased upon the addition of HSO₃^- ions while other metal ions didn’t show any changes in absorbance. Moreover, there was a color change observed from blue to colorless in naked eye. Upon fixing the excitation wavelength, probe had strong fluorescence emission at 685 nm but the strong emission was completely quenched upon the addition of HSO₃^- ions. Furthermore, sequential sensing studies were carried out for 39+HSO₃^- complexation against various analytes such as ROS, RNS and aminoacids. In colorimetric technique, probe 39 with HSO₃^- had weak absorbance and quenching of fluorescence emission but it was completely reversed upon the addition of H₂O₂ were the absorbance and fluorescence emission was restored to its natural position along with color change from colorless solution to blue in naked eye. The changes in absorbance and fluorescence emission was due to presence of HSO₃^- and H₂O₂, were upon the addition of HSO₃^- had 1,4-addition reaction that disappeared NIR emission but H₂O₂ addition underwent elimination reaction to reappear NIR emission to its original position. Another NIR fluorescent probe 40 was developed for the specific sensing of H₂O₂ for its application usages in wound healing process and clinical tests [89]. Before its spectroscopic examination, probe 40 was examined in presence and absence of H₂O₂ in absorbance spectra titration were 40 had absorption peak at 445 nm which was completely diminished in presence of H₂O₂ with a new absorption peak at 560 nm. Upon fixing the excitation wavelength for fluorimetric titration, probe 40 was evaluated against different analytes such as cations, anions, aminoacids, ROS and RNS. The probe 40 along with other analytes had weak fluorescence emission at 690 nm but presence of H₂O₂ alone had strong enhancement in fluorescence emission which clearly indicates that probe 40 with H₂O₂ recognition site could detect H₂O₂ at very fast sensitive against all other analytes. The fluorescence emission change was noted to be because of H₂O₂ presence which triggered the recognition site of fluorophore to enhance its emission selectively detecting H₂O₂. Furthermore, the two probes 39 and 40 was successfully utilized for its biological applications while probe 39 was proved to detect H₂O₂ in different food samples and also in MCF-7 living cells. Upon increasing the concentration of H₂O₂ in pre-treated cell lines with probe 39, firstly probe alone had weak fluorescence emission which was increased in presence of H₂O₂ that was visualized in red emission channel. Another probe 40 was used in detecting H₂O₂ against HACAT cell lines, wounded mouse and human diabetic foot. As discussed in spectroscopic titration techniques, probe 40 had weak emission at NIR region, so NIR emission was triggered using the addition of H₂O₂.

Figure 13. Structure of Chromene based fluorescent probes (39 & 40) for the detection of H₂O₂ ions and screening of H₂O₂ in wound healing process via mice model through probe 40. Reproduced with permission from Refs. [89], Copyright, American Chemical Society.

Figure 13. Structure of Chromene based fluorescent probes (39 & 40) for the detection of H₂O₂ ions and screening of H₂O₂ in wound healing process via mice model through probe 40. Reproduced with permission from Refs. [89], Copyright, American Chemical Society.

2.9. Benzothiazole Based Fluorescent Probes

Due to their distinctive photophysical characteristics and wide uses, benzothiazole-based fluorescent probes are indispensable in materials science, chemistry, and biology. Essential tools in the domains of analytical chemistry, biology, and materials science, the probes show great degrees of brightness and stability in fluorescence. These technologies' great selectivity helps to exactly identify certain biomolecules. Their flexibility also allows customizing for other kinds of experimental configurations. These probes help environmental monitoring and quality control initiatives in analytical chemistry by allowing the sensitive identification and quantification of analytes. These instruments are crucial for diagnostics and bioimaging in biomedicine as they provide real-time molecular visualizing of cellular events and disease indicators. Moreover, including these components into smart materials offers chances to design responsive systems able to change their characteristics in reaction to environmental cues. Constant research and development of benzothiazole-based fluorescent probes show significant improvement, thereby enabling developments in early illness diagnosis, focused treatments, and the creation of novel materials with particular use in mind. Using an AIE-ESIPT technique for the specific detection of HSO₃^- and sequential detection of H₂O₂, Lijun Tang and colleagues have created a new fluorescent probe labeled as 41 [90]. For use in cell imaging, the detecting technique was improved yet further. With absorbance measured at 600 nm and emission at 650 nm, probe 41 first showed notable absorbance and fluorescence emission features, ascribed to the interaction of AIE and ESIPT systems. Strong absorption and emission were seen when probe 41 was tested against many analytes including HSO₃^- ions. Absorbance was clearly reduced and fluorescence quenched, nevertheless, in the presence of HSO₃^- ions. Under normal lighting, this was followed by a clear color shift from blue to colorless; under UV-365nm irradiation, from red to colorless. Sequential selectivity against many molecules—including reactive oxygen species (ROS), reactive nitrogen species (RNS), and amino acids—was achieved using the complexation system probe 41+HSO₃^-. In presence of H₂O₂, the weak absorption and quenching of fluorescence emission were effectively induced. Increasing the concentration of H₂O₂ helped to return the absorbance and emission to their natural level, therefore reversing the color change. The 1,4 Michael addition reaction involving HSO₃^- and the retro- Michael addition reaction enabled by H₂O₂ may be responsible for the selective detection of HSO₃^- and sequential detection of H₂O₂ within the complexation system. Different spectroscopic methods verified the complexation mechanism even more. Designed for selective detection of H₂O₂ in live cells, a benzothiazole derivative-based fluorescent probe 42 was created [91]. Acting as an AIE probe, Probe 42 had poor fluorescence emission at 604 nm alone. On treatment with many analytes, only hydrogen peroxide (H₂O₂) showed a notable increase in fluorescence intensity; the other analytes did not clearly affect emission. The H₂O₂ presence was blamed for the variations in fluorescence emission. Probe 42 showed a "turn-off" fluorescence effect before H₂O₂ was added; this effect was then triggered in the presence of H₂O₂ and produced a "turn-on" fluorescence response. Successful development of a multifunctional fluorescent probe 43 for sequential H₂O₂ detection and SO₂ detection is reported. By means of the induced SO₂ and H₂O₂ procedures, the produced probe 43 was essentially used in the application process for both physiological and pathological circumstances connected to human illnesses [92]. Probe 43 first underwent analysis with respect to many analytes, including biological and environmental species. The probe showed a significant absorption peak at 610 nm, which dropped with SO₂ ion injection. By contrast, the probe showed a drop in fluorescence intensity at 750 nm and an increase at 510 nm when the excitation wavelength was fixed. H₂O₂ was sensed sequentially using a complexation method. Along with a rise in fluorescence intensity at 750 nm and a drop at 510 nm, a concentration of H₂O₂ significantly raised the absorption peak at 610 nm. This unequivocally shows that in the presence of H₂O₂ both fluorescence emission and absorbance was restored. The reversibility cycle showed that the probe, in the presence and absence of SO₂/H₂O₂, displayed restoration of absorbance and fluorescence emission following alternating addition up to six times, therefore suggesting its possible importance in biological processes. Designed and built by Lei Yang and colleagues, a near-infrared (NIR) fluorescent probe known as probe 44 could detect hydrogen peroxide (H₂O₂) using a fluorimetric titration method [93]. The efficacy of the probe in precisely tracking drug-induced liver damage was duly underlined. Originally in phase, the probe 44 had a modest absorption peak at 510 nm. H₂O₂ added helped to intensify this peak. Subsequently, the absorbance investigations let one ascertain the excitation wavelength for fluorimetric titration. At 772 nm, probe 44 showed no fluorescence emission signal during titration using many analytes. But when treated with H₂O₂, fluorescence intensity significantly increased, producing a 41-fold increase in emission. Fluorescence intensity consistently increased as the concentration of H₂O₂ with respect to probe 44 ranged from 0 to 100 µM. Furthermore absent from the complexation mechanism was interference from other analytes. The elimination of the boronic acid ester group in the presence of H₂O₂ produced the noted alterations in emission that resulted in the introduction of hydroxyl groups in probe 44. This change increases fluorescence emission by means of the intramolecular charge transfer (ICT) mechanism. Furthermore, all the probes investigated in this context were efficiently used for biological purposes involving many cell lines and animal tissues for the H₂O₂ detection. Probe 41 first showed intense red fluorescence when interacting with MCF-7 cells; however, this fluorescence decreased with increasing HSO₃^- concentration. Indicating that probe 41 may alternatively detect HSO₃^-/ H₂O₂ in live cells, the identical complexation system showed a recovery of H₂O₂ fluorescence intensity. Tested against two different cell lines, A549 and HepG2, Probe 42 showed modest fluorescence in the green channel. On higher concentrations of H₂O₂, however, fluorescence emission was clearly increasing. Fluorescence emission in the green channel significantly increased as concentration ranged from 5 µM to 100 µM. In photoacoustical and cell imaging, probe 43 was a multifarious detector for SO₂ and H₂O₂. Probe 43 showed considerable emission in the red channel when Na₂SO₃ was added, starting off with mild emission. Moreover, especially in studies involving the HepG2 cell line, increasing concentrations of H₂O₂ clearly resulted in a significant increase in fluorescence emission in the red channel. In the photoacoustic investigation, probe 43 was assessed in tumor-bearing and inflammatory animal models both with and without Na₂SO ₃/H₂O₂. Examining the probe 43 separately, we found a strong PA signal that was much reduced with NA₂SO₃'s addition and then recovered following H₂O₂ addition. Within the framework of the HepG2 cell line and drug-induced liver damage tissue, a near infrared probe 44 displaying high fluorescence emission was effectively assessed against H₂O₂. For probe 44, the experimental findings showed a high degree of performance in identifying H₂O₂ levels in the cell lines. Probe 44 showed great sensitivity to H₂O₂ in the investigation of samples treated for drug-induced liver damage, therefore indicating its possible use as a visual diagnostic and therapeutic drug screening tool (Figure 14).

Table 1. The fluorescent probes derived developed from various fluorophores for their detection towards H₂O₂ and their detection limit, detection technique, applications in animal tissues/foodstuffs/plants, and cell imaging.

Probe	Derivative	Detection method	Analyte	LOD	Animal tissues/ Food samples	Cell imaging	Reference
1	TPA	F	H2O2	0.141 μmol/L	Living normal tissues and tumor	HeLa	[50]
2	TPA	F	H2O2	0.25 mM	-	HeLa	[51]
3	TPA	C/F	H2O2	62 nM	Acute epilepsy & Chronic epilepsy mice	PC12	[52]
4	TPA	F	H2O2	0.34 μmol/L	Inflammation and ferroptosis mice	HepG2	[53]
5	TPA	F	H2O2	2.84 µM	Zebrafish	HepG2	[54]
6	TPE	F	H2O2	-	Tobacco leaves	Orin apple calli cells	[55]
7	TPE	C/F	H2O2	3.52 μM	Sugar	MCF-7	[56]
8	Quinoline	C/F	H2O2	44.6 nM	Zebrafish	HepG2	[57]
9	Quinoline	C/F	H2O2	23.08 nM	Potato tissues, Milk and Chicken wing	-	[58]
10	Quinoline	C/F	H2O2	0.87 μM	-	HeLa	[59]
11	Quinolinium	F	H2O2	0.17 μM	Diabetic mice	HeLa, HCT116 and 4T1 cells	[60]
12	Quinolinium	F	H2O2	210 nM	RA mice	HeLa	[61]
13	Quinolinium	C/F	H2O2	0.17 μM	Mice	SH-SY5Y	[62]
14	Quinolinium	F	H2O2	13 nM	CIRI rat	SH-SY5Y	[63]
15	Pinacol	C/F	H2O2	40.2 nM	Zebrafish	Raw 264.7	[64]
16	Pinacol	F	H2O2	0.003 μmol/L	-	HeLa	[65]
17	Pinacol	F	H2O2	-	Tumor-bearing mice	A375 cells	[66]
18	Pinacol	F	H2O2	49.74 nM	-	A549 cells	[67]
19	Pinacol	C/F	H2O2	386 nM & 16.8 nM	-	HeLa	[68]
20	Cinnamaldehyde	F	H2O2	-	-	HepG2	[69]
21	Hydroxybenzaldehyde	C/F	H2O2	1.02 mM	Zebrafish, mice and white wine, sugar samples	-	[70]
22	Carbaldehyde	C/F	H2O2	-	Cystitis mice	-	[71]
23	Benzaldehyde	F	H2O2	0.14 & 0.37 μm	Mice	HeLa	[72]
24	Carboxaldehyde	F	H2O2	112.6 nM	4T1 bearing tumor mice	4T1	[73]
25	Hydroxybenzaldehyde	F	H2O2	-	-	L-02 cells and HeLa, CT26 cells	[74]
26	Cinnamaldehyde	F	H2O2	0.36 μM	Tumor cell pyroptosis	4T1	[75]
27	Hemicyanine	F	H2O2	0.53 μM	HIRI and NAFL mice model	-	[76]
28	Hemicyanine	F	H2O2	0.38 μM	Mice tumor tissue	HeLa & A549 cells	[77]
29	Hemicyanine	C/F	H2O2	0.07 μM	Tomato leaves, stems, and roots	-	[78]
30	Hemicyanine	F	H2O2	0.14 µM	4T1 tumor-bearing mouse	4T1	[79]
31	Hemicyanine	C/F	H2O2	0.16 µM	PD-mice	SH-SY5Y	[80]
32	Hemicyanine	C/F	H2O2	0.361 mM	ApoE−/−/HF & ApoE−/−/HF/setanaxib group mice	LOX-1 & IL-1b	[81]
33	Dicyanoisophorone	F	H2O2	76 nM	Zebrafish	HepG2	[82]
34(i) (ii)	Dicyanoisophorone	F	H2O2	4.525 μM	Zebrafish	HeLa	[83]
35 (i) (ii)	Dicyanomethylene	F	H2O2	-	DU-145 tumor-bearing mice	MCF-10A, MCF-7, and DU-145 cells	[84]
36	Indolium	C/F	H2O2	0.59 μM	APAP-induced liver injury mouse	HeLa	[85]
37	Indole	F	H2O2	25.2 nM	Zebrafish	A459 cells	[86]
38	Trimethylindole	C/F	H2O2	1.82 × 10− 7 M	4T1 mice model	4T1 cells	[87]
39	Chromene	C/F	H2O2	2.157 μM	Liquor, Red wine, Sugar and Biscuit samples	MCF-7 cells	[88]
40	Chromene	C/F	H2O2	64 nM	Wounded mouse models	HeLa, HACAT	[89]
41	Benzothiazole	C/F	H2O2	0.152 μM	-	MCF-7 cells	[90]
42	Benzothiazole	F	H2O2	0.93 μM	-	A549 & Hep G2 cells	[91]
43	Benzopyrylium	C/F	H2O2	9.76 × 10− 7 M	Tumor-bearing mice	Hep G2 cells	[92]
44	Benzopyranonitrile	F	H2O2	17 nM	Liver injury mice	L02 and HeLa cells	[93]

Table 1. The fluorescent probes derived developed from various fluorophores for their detection towards H₂O₂ and their detection limit, detection technique, applications in animal tissues/foodstuffs/plants, and cell imaging.

Probe	Derivative	Detection method	Analyte	LOD	Animal tissues/ Food samples	Cell imaging	Reference
1	TPA	F	H2O2	0.141 μmol/L	Living normal tissues and tumor	HeLa	[50]
2	TPA	F	H2O2	0.25 mM	-	HeLa	[51]
3	TPA	C/F	H2O2	62 nM	Acute epilepsy & Chronic epilepsy mice	PC12	[52]
4	TPA	F	H2O2	0.34 μmol/L	Inflammation and ferroptosis mice	HepG2	[53]
5	TPA	F	H2O2	2.84 µM	Zebrafish	HepG2	[54]
6	TPE	F	H2O2	-	Tobacco leaves	Orin apple calli cells	[55]
7	TPE	C/F	H2O2	3.52 μM	Sugar	MCF-7	[56]
8	Quinoline	C/F	H2O2	44.6 nM	Zebrafish	HepG2	[57]
9	Quinoline	C/F	H2O2	23.08 nM	Potato tissues, Milk and Chicken wing	-	[58]
10	Quinoline	C/F	H2O2	0.87 μM	-	HeLa	[59]
11	Quinolinium	F	H2O2	0.17 μM	Diabetic mice	HeLa, HCT116 and 4T1 cells	[60]
12	Quinolinium	F	H2O2	210 nM	RA mice	HeLa	[61]
13	Quinolinium	C/F	H2O2	0.17 μM	Mice	SH-SY5Y	[62]
14	Quinolinium	F	H2O2	13 nM	CIRI rat	SH-SY5Y	[63]
15	Pinacol	C/F	H2O2	40.2 nM	Zebrafish	Raw 264.7	[64]
16	Pinacol	F	H2O2	0.003 μmol/L	-	HeLa	[65]
17	Pinacol	F	H2O2	-	Tumor-bearing mice	A375 cells	[66]
18	Pinacol	F	H2O2	49.74 nM	-	A549 cells	[67]
19	Pinacol	C/F	H2O2	386 nM & 16.8 nM	-	HeLa	[68]
20	Cinnamaldehyde	F	H2O2	-	-	HepG2	[69]
21	Hydroxybenzaldehyde	C/F	H2O2	1.02 mM	Zebrafish, mice and white wine, sugar samples	-	[70]
22	Carbaldehyde	C/F	H2O2	-	Cystitis mice	-	[71]
23	Benzaldehyde	F	H2O2	0.14 & 0.37 μm	Mice	HeLa	[72]
24	Carboxaldehyde	F	H2O2	112.6 nM	4T1 bearing tumor mice	4T1	[73]
25	Hydroxybenzaldehyde	F	H2O2	-	-	L-02 cells and HeLa, CT26 cells	[74]
26	Cinnamaldehyde	F	H2O2	0.36 μM	Tumor cell pyroptosis	4T1	[75]
27	Hemicyanine	F	H2O2	0.53 μM	HIRI and NAFL mice model	-	[76]
28	Hemicyanine	F	H2O2	0.38 μM	Mice tumor tissue	HeLa & A549 cells	[77]
29	Hemicyanine	C/F	H2O2	0.07 μM	Tomato leaves, stems, and roots	-	[78]
30	Hemicyanine	F	H2O2	0.14 µM	4T1 tumor-bearing mouse	4T1	[79]
31	Hemicyanine	C/F	H2O2	0.16 µM	PD-mice	SH-SY5Y	[80]
32	Hemicyanine	C/F	H2O2	0.361 mM	ApoE−/−/HF & ApoE−/−/HF/setanaxib group mice	LOX-1 & IL-1b	[81]
33	Dicyanoisophorone	F	H2O2	76 nM	Zebrafish	HepG2	[82]
34(i) (ii)	Dicyanoisophorone	F	H2O2	4.525 μM	Zebrafish	HeLa	[83]
35 (i) (ii)	Dicyanomethylene	F	H2O2	-	DU-145 tumor-bearing mice	MCF-10A, MCF-7, and DU-145 cells	[84]
36	Indolium	C/F	H2O2	0.59 μM	APAP-induced liver injury mouse	HeLa	[85]
37	Indole	F	H2O2	25.2 nM	Zebrafish	A459 cells	[86]
38	Trimethylindole	C/F	H2O2	1.82 × 10− 7 M	4T1 mice model	4T1 cells	[87]
39	Chromene	C/F	H2O2	2.157 μM	Liquor, Red wine, Sugar and Biscuit samples	MCF-7 cells	[88]
40	Chromene	C/F	H2O2	64 nM	Wounded mouse models	HeLa, HACAT	[89]
41	Benzothiazole	C/F	H2O2	0.152 μM	-	MCF-7 cells	[90]
42	Benzothiazole	F	H2O2	0.93 μM	-	A549 & Hep G2 cells	[91]
43	Benzopyrylium	C/F	H2O2	9.76 × 10− 7 M	Tumor-bearing mice	Hep G2 cells	[92]
44	Benzopyranonitrile	F	H2O2	17 nM	Liver injury mice	L02 and HeLa cells	[93]

3. Conclusions

The development of fluorescent probes employing near-infrared (NIR) and aggregation-induced emission (AIE) technologies is under more and more attention by researchers all over. These probes are very well-liked for their capacity to do both qualitative and quantitative studies of a broad spectrum of molecules, including cations, anions, amino acids, enzymes, and other physiologically important compounds. This review focuses on scholarly publications released between 2021-2025 that describe the design and synthesis of creative NIR/AIE-based fluorescent probes derived from various fluorophores for the detection of hydrogen peroxide (H₂O₂) using both colorimetric and fluorimetric spectroscopic techniques.

The methods of detection used are investigated in this study, thereby defining the processes of action and studying the many biological uses. All of the applications—cell imaging, animal tissue analysis, food sample testing, plant research—have an eye on H₂O₂. Furthermore, we underline the chemical approaches used to enhance the NIR emission characteristics of these fluorescent probes, particularly with regard to cancer-related disorders. This overview mainly addresses NIR-based fluorescent probes based on Förster resonance energy transfer (FRET) that function as intramolecular charge transfer (ICT)-dependent activatable fluorophores and systems. Innovations in this field usually combine quenching pairs with NIR-emitting systems, which are coordinated by interactions with metal ions, enzymes, peptides, or amino acids acting as targeting agents. Specifically discussed is the important function of NIR/AIE fluorescent probes in the selective detection of H₂O₂ in complicated biological settings. These probes show remarkable sensitivity for reactive nitrogen species (RNS), reactive oxygen species (ROS), and many other analytes, hence lowering the detection limit and increasing the quantum yield. Although many NIR/AIE fluorescent probes included in this study show amazing characteristics including excellent usage in numerous animal tissues, food samples, plants, and a variety of cell imaging applications across many cell lines, there is still much room for improvement. Still difficult are issues related to short emission wavelengths, naked eye vision, and interference from other analytes, all of which might influence diagnosis attempts linked with different disorders.

Moreover, the search of focused H₂O₂ probes for different organelles—including lysosomes and mitochondria—has presented many difficulties. By means of continuous efforts of the scientific community, it is expected that answers to these problems would progressively surface; producing more sophisticated and focused fluorescent H₂O₂ probes. High-performance probes are projected to provide rather powerful instruments for H₂O₂ detection in subcellular organelles. Eventually, in vivo applications made possible by this development will help to clarify cellular processes and disease causes.